1. Field of the Invention
The present invention relates generally to the formation of contrast agents containing cyclen-based chelates in order to produce multimodal images of a sample of cells.
2. Background Information
The American Cancer Society estimates that 16,800 new intracranial tumors were diagnosed in 1999, more than double the number of diagnosed cases of Hodgkin's disease, and over half the number of cases of melanoma. Moreover, in the same year, primary cancer of the central nervous system was the cause of death in approximately 13,100 people. Despite aggressive treatment strategies including surgical resection, irradiation and chemotherapy, most patients die from the disease, with median survival measured in months.
Patients with malignant gliomas 18-44 years old have a median survival rate of 107 weeks, whereas patients older than 65 survive just 23 weeks on average. Age, history of previous low-grade tumor, histologic composition of the tumor and treatments (i.e. radiation, surgery and chemotherapy) all affect prognosis. Yet, it is impossible to over-emphasize the importance imaging and detection techniques play in influencing the success in treating a variety of cancers.
Surgical therapy plays an important role in determining outcome for patients with primary brain tumors and other cancerous tumors. Gross total resection is associated with both longer survival and improved neurologic function. Therefore, every effort is made to remove as much of the tumor as possible. The degree to which a complete resection can be carried out in the brain is limited, however, by a number of factors unique to the central nervous system. One variable that directly influences the extent of resection is the difficulty of visually detecting differences between normal brain tissue and malignant tissue. Primary brain tumors are infiltrating lesions and the margins of the tumor are indistinct. For this reason, patients with malignant intrinsic brain tumors often undergo subtotal resection. Alternatively, patients may experience unexpected neurological morbidity if the resection is inadvertently carried into normal surrounding brain tissue. Additionally, current tumor imaging methods are limited because they image the tumor indirectly. Normally, imaging methods measure alterations in blood brain barrier permeability, detect edema or use non-specific MRI signal changes within the tumor and adjacent the brain. Thus it is difficult to identify radiation necrosis and inflammatory changes, as well as to assess response to therapy or to provide the capability of following disease progression. Furthermore, classical MRI contrast enhanced imaging fails to demarcate primary infiltrating glial tumors, with scans often indicating a discreet lesion border even though tumor cells typically extend several centimeters away. Because outcome is so closely linked to the extent of surgical resection, and the degree of resection is limited by difficulties in visually detecting tumors, there is a pressing need to develop new strategies to aid in intraoperative detection and imaging of brain cancer. The ideal imaging method for enhancing brain cancer detection would be tumor specific, non-invasive, provide real time intraoperative imaging and correlation with anatomic imaging (MRI), and define infiltrating tumor margins.
In patients where computed tomography (CT) or magnetic resonance imaging (MRI) are used to aid in providing “complete” resection of the tumor, the median survival period is 70 weeks. However, patients who undergo “only biopsy of the tumor” have a median survival rate of just 19 weeks. Even with this dramatic improvement, MRI and CT scans cannot be used to identify the outermost border of the malignant brain tumor. It has been repeatedly shown that areas that appear “normal” during surgery contain tumor cells. While contrast enhanced MRI scans often suggest the presence of a discreet border to the lesion, brain tumors are widely infiltrative with tumor cells typically extending microscopically several centimeters away from the obvious area of the disease. Current imaging methods do not adequately assess the extent of disease or differentiate between viable tumor infiltration, radiation necrosis, or inflammatory changes. For these reasons, current imaging modalities are not ideal for following disease progression or assessing response to therapy. If it were possible to perform guided resection using a fluorescent contrast agent to image brain cancer cells on a microscopic scale in the operating room in combination with macroscopic scale MRI, the long-term survival rate would significantly improve.
Recent discoveries in molecular imaging play a vital role in the early detection, diagnosis, and treatment of disease, as well as in the study of biological and biochemical mechanisms, immunology, and neuroscience. Since current molecular-level technologies primarily focus on in-vitro methods, it is crucial to develop imaging methodologies that have high spatial and temporal resolution in-vivo and spectroscopy techniques for imaging at the cellular or molecular scales. Presently, in-vivo detection and surgical resection means are limited to either macroscopic gross visualization, white-light endoscopy or white-light microscopic visualization. The visual assessment of any tissue depends on many factors, including the experience of the clinician, his/her ability to identify the suspect lesions and their resection or biopsy skill. The visual cues, such as signal to noise ratio (S/N), for the determination of a pathologic state are small or low, especially for brain cancer which tends to infiltrate normal tissue. When the contrast for disease is low as it is for small foci of cancer cells, it makes surgical resection problematic. These techniques tend to miss disease at its earliest stage because of low concentrations of the endogenous chromophores leading to contrast between normal tissue and disease. The need for better contrast agents to assist in identifying disease is not limited to the brain. For example, patients undergoing back-to-back colonoscopies, performed by an experienced colonoscopist, may have as many as 15% to 24% of their neoplastic polyps smaller than 1 cm overlooked. Furthermore, up to 6% of larger polyps may escape detection as well. In short, clinical outcomes are dependent on the design of new contrast agents and imaging methodologies. Clearly, there is a significant need for the enhancement of disease detection to improve the clinical outcomes.
Recent advances in optical imaging, high spatial resolution MR, and nuclear imaging play an important role in obtaining molecular information. Early-stage disease detection, evaluation of molecular markers for therapy assessment, and imaging of gene expression or protein levels are just a few of the applications immediately available. High-affinity ligands developed rationally, combinatorially, or by chance must have the ability to reach the intended target at sufficient concentration and for a sufficient length of time to be detectable in-vivo. Through the use of compounds with multiple signatures, this goal is more readily achieved.
Although MRI remains the hallmark imaging modality for examining many types of cancers, MRI does not provide a clinician with real-time intra-operative maps once surgery commences. Thus, new MR contrast agents that can also offer real time intra-operative fluorescence visualization of disease need to be developed in order to take full advantage of conventional MRI examination. While there are numerous exogenous, topical or injectable agents being used to aid in the demarcating of normal tissue from disease tissue, including acetic acid, indocyanine green (ICG), methylene and toluidine blue, and 5-aminolevulinic acid (ALA), none of these agents can be used as an MRI agent. These agents are also restricted in use due to tissue toxicity, low specificity, and/or long manifestation times, with contrast primarily based on differential permeability of the agent between normal and diseased tissue. Generally they provide only modest diagnostic utility, primarily because of an inherent lack of specificity.
Several research groups have investigated laser-induced fluorescence spectroscopy (LIFS) of endogenous fluorophores as a potential tool for tissue diagnostics, targeting cardiovascular and oncological applications. Pertinent reviews in the field of Laser-Induced Fluorescence Spectroscopy (LIFS) for tissue diagnostic applications, and in particular, oncological applications have been presented. Evidence exists that tissue staging can be accomplished allowing transformation from dysplasia to cancer to be quantified. All tissue contains fluorophores or endogenous chromophores that absorb light and subsequently emit light at a longer wavelength. Nicotinamide adenine dinucleotide (NAD[H]), flavins, collagen, and elastin are commonly known tissue fluorophores. It is currently believed that autofluorescence primarily detects changes in concentration or distribution of these components. As normal tissue becomes dysplastic the concentration or distribution of these endogenous fluorophores changes thus leading to a detectable change in the resulting fluorescent spectrum. These changes are wavelength dependent and correlate with changes in histology. These techniques, while showing great promise, still suffer from relatively low S/N stemming from small changes in concentration of the solutes detected in early disease and the large background arising from other fluorophores, scattering, and reflected light. Combining these technical advances with new contrast agents will advance the field of molecular imaging.
Moreover, while steady-state LIFS techniques have been extensively investigated and are currently routinely used in research clinics for characterization of endogenous and exogenous fluorescence, only a few research groups have explored the TR-LIFS techniques for diagnostics. In this regard, both time- and frequency-domain time-resolved instrumentation have been employed. This early work suggests that time-resolved fluorescence spectroscopy improves the specificity of fluorescence measurements in tissue and enhances the ability of LIFS to characterize tissue composition. The use of time-resolved fluorescence approach for tissue characterization is suitable for several reasons. Time-resolved fluorescence measurement a) can resolve the spectral overlap of endogenous fluorophores in tissue (main limitation of LIFS steady-state), b) is independent of fluorescence emission intensity as long as the signal to noise is commensurable, thus independent to the presence of the endogenous chromophores in tissue (hemoglobin) or to excitation-collection geometry (optical assembly), and c) is sensitive to microenvironmental parameters in tissue (pH, enzymatic activity, temperature), thus various tissue parameters can be monitored. Cellular level detection and evaluation of glioma could be facilitated by combining the power of time-resolved spectroscopic imaging with contrast agents.
Another example of contrast enhancement agents or site-directed chemical agents that have seen recent success is the PhotoDynamic Therapy (PDT) class of markers. While these types of markers have shown promise in a diagnostic setting, there are limitations. Long delays for accumulation in tumors, prolonged photosensitization of skin, and phototoxicity of tissues being imaged are some examples of these limitations. Preclinical studies in rat brain-tumor model with hematoporphyrin derivative (HpD) demonstrated an increase in the HpD fluorescence in the brain tumor relative to the adjacent normal tissue. Second generation photosensitizers such as chloro-aluminium phthalocyanine have been shown to increase the accuracy with which rat brain tumor margins can be defined during resection in-vivo. More recently, 5-Aminolevulinic acid-(ALA) induced porphyrin (PpIX) fluorescence has been used in clinical studies. The results suggest that ALA-induced PpIX may label malignant gliomas and enhance the tumor removal. However, to date, their efficiency in intraoperative detection of tumor margins has been limited.
Improved site-directed delivery of contrast agents can be accomplished when a carrier molecules or ligand specific for a receptor is conjugated to some signaling species. In general the result is enhanced accumulation or association of this ligand with or in a certain type of cell, leading to high detection specificity when using of fluorescence imaging. For example, it has recently been demonstrated that implanted pancreatic acinar tumors, that over express the somatostatin receptor, could be imaged using a DOTA and a Indocyanine Green (ICG) conjugate. Another example of site directed contrast enhanced imaging is the use of folate to target several different kinds of cancer cells that are known to upregulate a receptor for this complex. One drawback of this approach to cancer detection is that the chemistry required for conjugation can often be quite complicated or require great synthetic skill.
Pyclen-based lanthanide chelates can be used as exogenous markers for epithelial carcinoma. Pyclen-based terbium chelate having the following chemical structure have been used to detect chemically induced colon cancers in the Sprague Dawley rat. As shown below, lanthanide chelates can be derived from the following ligands: 3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(15),11,13-triene (PCTMB), 3,6,9-tris(methylene phosphonic acid)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(13),11,13
triene (PCTMP) and N,N′-bis(methylene phosphonic acid)-2,11-diaza[3.3]-(2,6)pyridinophane (BP2P). The molecule, Tb-[N-(2-pryidylmethyl)-N′,N″,N′″-tris(methylenephosphonic acid butyl ester)-1,4,7,10 tetraazacyclododecane] or Tb-PCTMB has excellent fluorescent properties, good specificity, and low toxicity. Preliminary work indicates that Tb-PCTMB can be used as an exogenous marker for dysplastic tissues with sensitivity as high as 94.7%. Using the bright green fluorescence from a tributyl ester Tb3+ chelate with a tethered antenna, and subsequent histopathology, it is possible to detect squamous cell carcinoma lesions in the Syrian Hamster Cheek Pouch which are not visible by standard white light imaging. However, a major concern with using these pyclen-based lanthanide chelates is that the harvesting moiety, pyridine, requires high energy UV light (270 nm) to excite the lanthanide, which can be harmful to tissues. Therefore, a new class of chelates that does not require high energy UV light yet still retains a high degree of specificity and sensitivity is desirable.
When used as molecular imaging agents, contrast agents having conjugated complexes have the potential for high discrimination between histologically dissimilar (i.e. normal vs. diseased) tissues on the molecular level. In particular, mitochondrial function, specifically the over-expression of the peripheral benzodiazepine receptor (PBR) in cancerous tissue, has been suggested as an effective target with which to direct contrast agents for the identification of disease, especially cancers of the CNS. Although PBRs are widely expressed throughout the body in all steroid-producing tissues, their density in the CNS is primarily limited to the ependymal cells and glia. This 18 kDa protein is associated with many biological functions including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogensis and apoptosis, all processes that would be amplified in immortalized, rapidly proliferating tumor cells. The exploitation of PBR over expression has been shown to be a viable targeting technique for numerous times. A known high affinity PBR ligand PK11195 has been used both as an in-vitra and in-vivo PET agent for visualizing both human and rat gliomblastoma, and as a targeted therapeutic agent when conjugated to the drug gemcitabine. It has also been shown that a PBR ligand which is closely related to PK-11195, 7-Nitro-2,1,3-benzoxadiazol-4-yl, could be conjugated to a fluorescent label, 2-Phenylindole-3-acetamide, retaining the ability label PBR in glioma cell lines. While this compound is attractive, it provides only an optical signature and has not been conjugated to the most attractive PBR ligand, PK-11195.
For various reasons, including non-ideal interstitial solute transport properties and complicated pharmokinetics, the approach of using high-affinity vector molecules as contrast agents has been limited by poor specificity when used in vivo. This has not been the case when the isoquinoline, PK-11195, has been used as a ligand to target the peripheral-type benzodiazepine, a PBR ligand that labels glioma cells. Human glial tumors have been shown to display a high density of peripheral benzodiazepine binding (PBR) sites. The presence of such high concentrations of specific PBR receptors on glial tumors suggests that human primary brain tumors could be imaged and diagnosed using PBR ligands. Recent reports have demonstrated the feasibility of this hypothesis and have identified the peripheral benzodiazepine receptor ligand PK-11195 as an excellent candidate molecule. Intravenously administered 3H-PK-11195 has been shown to selectively label glioma cells in rat brains studied in-vitro and in-vivo. Moreover, autoradiograms of postmortem brain sections containing glioma revealed that 3H-PK-11195 bound specifically to intact tumor cells and not to cells of normal cortex or necrotic areas of the tumor. A clinical positron emission tomography (PET) study was carried out with C11PK-11195 and demonstrated that saturable, high-affinity binding of the ligand corresponded directly to the tumor. Taken together, these findings provide support for the use of PK-11195 to image human gliomas. However, these studies have not been fully explored nor demonstrate the potential use of PK-11195 as a vehicle for contrast enhancement in brain cancer imaging. In addition, to imaging brain cancer, PK11195 may be useful in the identification of other forms of cancer. For example, breast cancer cells over-express substantial quantities of PBR as well.
At this time, there are no specific markers for neoplastic glial cells, thus allowing the unambiguous discrimination of human brain tumors from normal tissue. While there is a marker for glial cells called glial fibrillary acidic protein (GFAP), it is also present in reactive and normal glial cells. Furthermore, GFAP is inconsistently detectable in glioma cells. A sensitive and specific marker for neoplastic glial (glioma) cells would be of particular assistance in the identification of low cellularity infiltrating glioma cells amidst reactive glia. It may also have utility as a marker for distinguishing gliomas from other malignancies.
Current MRI modalities significantly improve patient diagnosis when tumor tissue is clearly distinguishable from normal tissue. In an attempt to fully identify a tumor in its entirety, clinicians infuse patients with a Gd3+ containing contrast agent that exploits the varying degrees of angiogenesis between normal and diseased tissue. An effective method for increasing MR contrast in-vivo is to increase the relaxivity of the contrast agent, thus yielding greater differentiation between diseased and normal tissue with a T1 weighted image, facilitating smaller agent doses to be administered reducing toxicity. Several groups have shown that by attaching Gd3+ chelating agents to macromolecules the relaxivity of the combined contrast agent can increase substantially. Additionally by attaching several molecules of the contrast agent to polylysine (PL) and/or poly(ethylene glycol) (PEG), relaxivities in the 2500 mM−1s−1 range can be obtained. These relaxivities are three orders of magnitude better than commercially available MR contrast agents, because the greater size and amount of Gd3+ present. It has recently been shown that paramagnetic ions can also be bound to proteins to yield an increase in relaxivity. Alternately, MR contrast can be improved by more effectively targeting the delivery of the agent. Iron complexes have been investigated, but as single entities they show no real enhancement over those of Gd(III).
Lanthanides containing compounds have also been used as temperature sensors. The optical fiber community has exploited this temperature dependent fluorescence of rare earths for some time relying on changes in the up-converted ionic fluorescent intensity or fluctuations in the ionic fluorescence lifetime. Current detection methodologies rely primarily on changes in the up-converted ionic fluorescent intensity or fluctuations in the ionic fluorescence lifetime. Typical fiber optic temperature sensors are temperature sensitive in the range from 20° C. to 140° C., with sensitivities on the order of 1×10−2 Δ° C.1. A new class of compounds that provides both improved tissue imaging, which also has the ability to exploit the temperature sensitivity of lanthanide series ions would be desirable.